The identification, analysis and monitoring of biological compounds, most importantly proteins, polysaccharides and the like, has become increasingly important for research and industrial applications, as well as health care fields, demographic analysis and the like. Conventionally, biological analyte detection systems are based on detection of an antibody binding to the suspected antigen in the analyte, or vice-versa, and somehow determining the (1) event of binding and (2) Mount of binding between antigen and antibody occurring. In general, the specific binding pair (wherein the antibody is specific for the antigen, and will not bind to non-antigen mimics) is referred to as an antibody-antigen complex, or immunocomplex. If the occurrence of binding per se, is all that is measured, the assay is qualitative in nature. Increasingly, however, quantitative measuring, i.e., the amount of binding, is of concern. Such quantitative analysis requires enhanced sensitivity as well specificity.
One dominant form of immunoassay providing information of this type is the sandwich ELISA, wherein an antibody, bound to a stable support surface, is admixed with the analyte, and antigen present in the analyte is bound to the previously bound antibody. The surface binding the antibody is then washed to remove debris. Thereafter, the bound antigen is contacted with a preparation of another antibody, this second antibody bearing a reporter molecule or label of some sort which can be detected. The second antibody will bind to the exposed and bound antigen, and after a second washing, the Mount of second antibody bound can be detected, by determining the label concentration. The sandwich assays can be competitive or non-competitive in form. They require, however, at a minimum, the preparation of two different antibodies, several wash steps, and introduce a series of reactions which must be detected, and thus, the reliability and sensitivity of the assay is dependent upon repeated complexing events.
In response to these difficulties, a variety of more direct assays to detect the formation of the immunocomplex have been developed. Thus, a variety of assays have been developed to measure the specific binding of an antigen-antibody specific binding pair, by attaching a fluorescent label, such as fhorescence to one element of the binding pair, and attaching a quenching particle, that is, a molecule or molecular fragment which quenches the fhorescent activity of the fhorescent label, to the other binding pair. The fluorescence of the fluorescent-label-bound element is monitored as it is added to the second element. The binding of the antibody and the antigen brings the quenching moiety in close proximity to the fluorescent label, extinguishing or reducing the degree of fluorescence. Thus, a reduction in detected fhorescence is indicative of binding, and the degree of reduction is indicative of the degree or amount of binding. U.S. Pat. Nos. 3,996,345, Ullman et al, 4,650,770, LiU et al, 4,351,760, Khanna, 3,998,943, Ullman and 4,806,488, Berger et al, all describe variations on this theme. In an alternative, but related format, one of the antibody or antigen ligand is labeled with a reporter molecule, and the complexing of the antibody and antigen inhibits the attack on the label, by some subsequently added agent. Thus, U.S. Pat. No. 4,208,479, Zuk et al, adds a modifying agent after formation of the immunocomplex, which modifying agent may be light. Unbound label will be altered by the modifying agent, to fluoresce at a different wave length, or give off a different degree of fluorescence, allowing distinction between bound and unbound label. Similarly, U.S. Pat. No. 4,816,419, Halfman, calls for the addition of a surfactant to a immunocomplex-containing preparation. Either the antigen or the antibody of the complex is labeled with a fluorescent dye. The surfactant forms micelles awhich sequester unbound label material, thereby quenching the label, while bound label will continue to fluoresce. In a third alternative, various dye sequences, or selective dyes, are used, the spectral absorbance of the dye shifting upon formation of the immunocomplex. Assays of this type are addressed in U.S. Pat. No. 4,568,647, Sanford, and U.S. Pat. No. 4,166,105, Hirschfeld.
All of the above direct assays are characterized by the need to interact the fluorescent label with a second molecule or moiety of some type, which alters the degree of fhorescence. This requires additional complexing, which may be difficult when the antigen is not readily identifiable. Further, most of these sequences require additional washes, where an agent is added to the complex, to alter the characteristics of the fluorescence.
Accordingly, it remains a goal of those of ordinary skill in the art to provide a direct, homogeneous assay for antigen-antibody specific binding, which allows both qualitative and quantitative determination of the presence of the antigen-antibody complex.